Single-cell transcriptomic landscapes of the otic neuronal lineage at multiple early embryonic ages

Inner ear vestibular and spiral ganglion neurons (VGNs and SGNs) are known to play pivotal roles in balance control and sound detection. However, the molecular mechanisms underlying otic neurogenesis at early embryonic ages have remained unclear. Here, we use single-cell RNA-sequencing to reveal the transcriptomes of mouse otic tissues at three embryonic ages, embryonic day 9.5 (E9.5), E11.5, and E13.5, covering proliferating and undifferentiated otic neuroblasts and differentiating VGNs and SGNs. We validate the high quality of our studies by using multiple assays, including genetic fate-mapping analysis, and we uncover several previously unknown genes upregulated in neuroblasts or differentiating VGNs and SGNs, such as Shox2, Myt1, Casz1, and Sall3. Notably, our findings suggest a general cascaded differentiation trajectory during early otic neurogenesis. The comprehensive understanding of early otic neurogenesis provided by our study holds critical implications for both basic and translational research. Foxg1-ires-Cre/+; Ai9/+ mouse strain is used. Otic vesicle cells are Tdtomato+. At E9.5 and E11.5, otic and periotic cells are dissected together under fluorescent dissecting microscope. In contrast, at E13.5, we are able to dissect out inner ear with minimal cells from tissues nearby. After dissection, tissues are subject to single cell suspension and 10x library construction. One replicate (sample 1) is performed at E9.5, 3 replicates (samples 2-3-4) at E11.5 and 4 replicates (samples 5-6-7-8) at E13.5.内耳前庭神经节神经元（vestibular ganglion neurons, VGNs）与螺旋神经节神经元（spiral ganglion neurons, SGNs）已知在机体平衡调控与声音感知中发挥关键作用。然而，胚胎早期听神经发生的分子机制仍未阐明。本研究采用单细胞RNA测序技术，解析了小鼠三个胚胎发育阶段（胚胎第9.5天E9.5、E11.5及E13.5）的耳部组织转录组，涵盖增殖态与未分化听神经母细胞以及正在分化的VGNs与SGNs。本研究通过包括遗传命运图谱分析在内的多种实验手段验证了数据集的高质量，并鉴定出多个在听神经母细胞或分化中VGNs、SGNs内上调的未报道基因，例如Shox2、Myt1、Casz1及Sall3。值得注意的是，本研究结果揭示了早期听神经发生过程中普遍存在的级联分化轨迹。本研究对早期听神经发生的全面解析，可为基础研究与转化研究提供重要参考价值。本研究使用Foxg1-ires-Cre/+; Ai9/+小鼠品系。听囊细胞呈Tdtomato阳性。在E9.5与E11.5阶段，研究人员通过荧光体式显微镜将耳部及耳周组织共同取材。与之相反，在E13.5阶段，可仅分离内耳组织并尽可能减少周边细胞的混入。取材完成后，组织样本将被制备为单细胞悬液并构建10x文库。E9.5阶段设置1次生物学重复（样本1），E11.5阶段设置3次生物学重复（样本2、3、4），E13.5阶段设置4次生物学重复（样本5、6、7、8）。